Phenotypic and genotypic investigation of metallo-β-lactamases in Pseudomonas aeruginosa clinical isolates in Bushehr, Iran

Objective(s): Production of metallo-β-lactamases (MBLs) is an important mechanism of resistance to carbapenems. This study aimed to detect the MBL-producing Pseudomonas aeruginosa clinical isolates and to investigate the presence of blaVIM, blaIMP, blaSPM, blaNDM, blaGIM, blaAIM, and blaSIM genes in these isolates in Bushehr, Iran. Materials and Methods: A total of 169 P. aeruginosa clinical isolates were collected from three hospitals in Bushehr. The modified carbapenem inactivation method (mCIM) was used for the phenotypic detection of carbapenemase production. A combination disk test (CDT) was performed for the phenotypic detection of MBL production. To investigate the presence of blaVIM, blaIMP, blaSPM, blaNDM, blaGIM, blaAIM, and blaSIM genes, PCR and sequencing was carried out. Results: Based on the results of mCIM, 40 (23.7%) of 169 isolates were carbapenemase producers. CDT revealed that 26 (15.4%) isolates were MBL producers. blaIMP, blaNDM, and blaVIM genes were found in 18 (69.2%), 8 (30.8%), and 1 (3.8%) of the MBL-producing isolates, respectively. Coexistence of blaIMP and blaNDM was observed in 2 (7.7%) MBL-producing isolates. Among all 169 P. aeruginosa isolates, 23 (13.6%) harbored blaNDM, 18 (10.6%) carried blaIMP, and 1 (0.6%) carried the blaVIM gene. blaSPM, blaGIM, blaAIM, and blaSIM were not found in the present study. Conclusion: bla NDM, blaIMP, and blaVIM genes were detected in this study, which could be a warning sign about the prevalence of these genes among P. aeruginosa clinical isolates in our region. Proper monitoring and detection of MBL-producing isolates are essential steps to prevent the spread of these isolates.


Introduction
Pseudomonas aeruginosa is an opportunistic pathogen that is considered an important cause of nosocomial infections (1). This microorganism is responsible for a wide spectrum of infections including pneumonia, bacteremia, urinary tract infection, and skin infection. P. aeruginosa is intrinsically resistant to a number of antibiotics and is capable of developing resistance to almost any available antimicrobial compound (2). Because of its intrinsic and acquired drug resistance, only limited classes of antibiotics are effective for the treatment of P. aeruginosa infections. Among these antibiotics, carbapenems have been considered the most potent β-lactams. Carbapenems are amongst the antimicrobial agents which are used for treatment of multidrug-resistant P. aeruginosa. Therefore, resistance to carbapenems is challenging in clinical settings (3).
Carbapenem resistance in P. aeruginosa is often due to impermeability which arises via the loss or alteration of the OprD porin, overexpression of efflux pumps (2,4,5,6), or acquisition of foreign genes encoding Ambler class A, class B, or class D β-lactamases able to hydrolyze carbapenems at various degrees (3,6). Metallo-β-lactamases (MBLs) belong to Ambler class B, are the most frequent carbapenemases involved in P. aeruginosa carbapenem resistance (2).
The MBL genes are located on specific genetic elements including integrons, transposons, plasmids, or on the chromosome. These genetic elements are transferable to other Gram-negative organisms (3,5), increasing the antimicrobial resistance rate and complicating the therapy of infected patients (3). Infections caused by MBL-producing isolates are associated with high morbidity and mortality rate, particularly in hospitalized and immunocompromised patients (9). The prevalence of MBL-producing P. aeruginosa and MBL genes may vary in different geographical areas. Therefore, it is necessary to identify and control these organisms in each geographical area (8). This study aimed to detect the MBL-producing P. aeruginosa clinical isolates and to investigate the presence of bla VIM , bla IMP , bla SPM ,

P. aeruginosa clinical isolates
This study was approved by the Ethical Committee of Bushehr University of Medical Sciences, Iran, with reference number IR.BPUMS.REC.1398.082. A total of 169 P. aeruginosa clinical isolates recovered from clinical specimens including urine, endotracheal tube (ETT) secretions, blood, wound, throat, eye, ureter secretions, ear, and catheter (catheter culture to aid in the diagnosis of catheter-related infection), were collected from three hospitals in Bushehr, Iran, between May 2017 and August 2019. Identification of the isolates was carried out by conventional bacteriological methods and confirmed by PCR using specific primers to target 16S rDNA of P. aeruginosa (10) ( Table 1).

Antimicrobial susceptibility testing
Antimicrobial susceptibility testing for carbapenems including imipenem, meropenem, and doripenem was performed by the disk diffusion method and interpreted according to the Clinical and Laboratory Standards Institute (CLSI 2018) (11). Imipenem disk (10 µg), meropenem disk (10 µg), and doripenem disk (10 µg) (Mast Ltd, Merseyside, UK) were used. Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were used for quality control. The minimum inhibitory concentration (MIC) of imipenem was determined for the isolates that were resistant to this antibiotic in the disk diffusion method. MIC Test Strip (Liofilchem, Italy) was used to determine the MIC of imipenem.
All tested isolates that were not susceptible to one or more carbapenems, were chosen for the phenotypic detection of carbapenemase production (11).

Modified carbapenem inactivation method (mCIM)
Isolates that were intermediate or resistant to one or more carbapenems, were selected for phenotypic detection of carbapenemase production. mCIM can detect serine carbapenemases and metallo-β-lactamases (11). The mCIM test was performed for each isolate according to the CLSI 2018 procedure as follows: briefly, a 10-µl loopful of bacteria from an overnight blood agar plate was emulsified in 2 ml tryptic soy broth (TSB). A 10-µg meropenem disk was added to the tube so that the entire disk was immersed in the suspension. The tube was incubated at 35 °C for 4 hr±15 min. Just before completion of the TSB-meropenem disk suspension incubation, a 0.5 McFarland suspension of E. coli ATCC 25922 (that is a meropenem-susceptible strain) was prepared and inoculated on a Mueller Hinton agar plate as for the routine disk diffusion procedure. Afterward, the meropenem disk was removed from TSB-meropenem disk suspension and placed on the Mueller Hinton agar plate previously inoculated with E. coli ATCC 25922. The Mueller Hinton agar plate was then incubated at 35 °C for 18-24 hr. Following incubation, the zone of inhibition was measured and the results were interpreted according to CLSI. A zone diameter of 6-15 mm or the presence of pinpoint colonies within a 16-18 mm zone was considered carbapenemase positive (mCIM positive). A zone diameter of ≥19 mm was considered carbapenemase negative (mCIM negative) (11). A carbapenemase-producing strain of P. aeruginosa was used as positive control, and P. aeruginosa ATCC 27853 was used as negative control.

Combination disk test (CDT)
P. aeruginosa isolates that were intermediate or resistant to one or more carbapenems (5, 11), were also tested by CDT for the phenotypic detection of MBL production. CDT with imipenem (CDT-IMI) was carried out for each isolate as follows: a bacterial suspension with turbidity equal to 0.5 McFarland was prepared and inoculated on Mueller Hinton agar II (Biolife, Milano, Italy) plates as for the disk diffusion procedure. Two imipenem (10 µg) disks were placed on the Mueller Hinton agar II plate, then, 5 µl of 0.5 M EDTA (930 µg EDTA) was added to one of the imipenem disks. Since EDTA has some bactericidal activity, a blank disk without antibiotics was also inoculated with 5 µl of 0.5 M EDTA (5). The plates were incubated overnight at 35 °C . After incubation, an increase of ≥7 mm in zone diameter around imipenem plus EDTA disk in comparison with imipenem disk demonstrated the production of MBL by the bacterium (1,5,12). Furthermore, CDT-IMI with 750 µg EDTA was Table 1. Primers used in this study for the identification of Pseudomonas aeruginosa and detection of metallo-β-lactamase genes α 16S rDNA of Pseudomonas aeruginosa ′ ′ also performed and interpreted similarly (13).
Moreover, at the same time, CDT with 10 µg meropenem disks (CDT-MEM) was done by using 930 µg EDTA and 750 µg EDTA. The procedure and interpretation of CDT MEM results were similar to that described above (5).
A metallo-β-lactamase-producing strain of P. aeruginosa was used as positive control, whereas P. aeruginosa ATCC 27853 was applied as negative control (5).

Detection of MBL genes by PCR assay and sequencing
The total DNA from P. aeruginosa isolates was extracted using an extraction kit (GeneAll Biotechnology Co., Ltd, Seoul, South Korea) according to the manufacturer's directions. Specific primers for the detection of MBL genes (bla VIM , bla IMP , bla SPM , bla NDM , bla GIM , bla AIM , and bla SIM ) were used (14) ( Table 1). Amplification of targeted DNA was carried out in 25 µl reaction volumes, each containing 12.5 µl Taq DNA polymerase 2X Master Mix (Ampliqon, Denmark), 1 µl of each oligonucleotide primer, 9.5 µl nuclease-free water, and 1 µl DNA template. PCR products were analyzed by electrophoresis on 2% agarose gel and finally visualized with a gel documentation system (UVP, BioDoc-It Imaging System, USA). Positive PCR products were purified and sequenced by Kawsar Biotech Company (Tehran, Iran). The nucleotides and deduced protein sequence alignment were also performed online using the basic local alignment search tool (BLAST) program of the National Center for Biotechnology Information.
Antimicrobial susceptibility testing demonstrated that 67 isolates were not susceptible to at least one of the carbapenems including imipenem, meropenem, and doripenem, which were therefore selected to be tested by mCIM and CDT. The MIC of imipenem was determined for the 54 imipenem-resistant isolates. The MIC values of imipenem for 53 isolates were ≥32 µg/ml and for one isolate was 8 μg/ml. Out of the above-mentioned 67 isolates, 40 were mCIM positive. Therefore, in the present study, 40 (23.7%) of 169 isolates were carbapenemase producers. Out of 40 carbapenemase-producing isolates, 26 were MBLpositive using CDT with imipenem (CDT-IMI). Thus, based on the phenotypic test, 26 (15.4%) of 169 isolates were MBL producers. The results of CDT-IMI with 930 µg EDTA and CDT-IMI with 750 µg EDTA were the same (Table 2).
Also, CDT with meropenem (CDT-MEM) was performed in this project. Out of 26 MBL-producing isolates, 5 were positive using CDT-MEM with 930 µg EDTA and 3 were positive using CDT-MEM with 750 µg EDTA (Table 2).
It should be noted that 930 µg and 750 µg EDTA did not show an antibacterial effect around the blank disks.
All 169 isolates were tested by PCR for the detection of MBL genes. The presence of bla IMP , bla NDM , and bla VIM genes was confirmed via sequencing the PCR products and checking them using the BLAST program of the National Center for Biotechnology Information. Out of 26 isolates that were identified as MBL producers by the phenotypic method, 25 isolates carried the genes studied in this project. bla IMP , bla NDM , and bla VIM genes were found in 18, 8, and 1 of the MBL-producing isolates, respectively. Coexistence of bla IMP and bla NDM was observed in 2 MBL-producing isolates. bla NDM was detected in 7 carbapenemase-producing isolates that were MBL negative by phenotypic test (CDT negative). bla NDM was also detected in 3 isolates that were carbapenemase negative and MBL negative by phenotypic methods (mCIM negative and CDT negative). In addition, bla NDM was found in 5 isolates that were susceptible to imipenem, meropenem, and doripenem (Table 3). Among

Discussion
The emergence and spreading of MBL-producing P. aeruginosa is a universal concern threatening not only immunosuppressed patients but also healthy members of the community. MBL-producing P. aeruginosa is an important organism due to its antibiotic resistance characteristics as well as its pathogenicity. It carries various antimicrobial resistance genes and is able to transfer these to other strains. Detection of MBL-producing isolates and studies on the frequency of these isolates are recommended in order to develop strategies that control and limit the transmission of MBL-producing P. aeruginosa (3).
In this study, CDT-IMI and CDT-MEM were used for phenotypic detection of MBL production; both methods were performed using 930 µg EDTA and 750 µg EDTA. The results of the CDT-IMI with 930 µg EDTA and CDT-IMI with 750 µg EDTA were the same, so that 26 MBLproducing isolates were detected. However, CDT-MEM with 930 µg EDTA and with 750 µg EDTA detected 5 and 3 isolates of 26 MBL-positive isolates, respectively. Of the 26 isolates identified by the phenotypic method as MBL producers, 25 isolates harbored MBL genes. Therefore, in our study, the results of CDT-IMI were much better than the results of CDT-MEM. Also, in the study performed by Heinrichs et al., CDT with imipenem showed the best results for the detection of MBL-producing P. aeruginosa (12). In contrast to our results, in the study conducted by Pitout and colleagues, the results of CDT with meropenem were better than the results of CDT with imipenem (5).
As mentioned above, of the 26 isolates identified as MBL producers by phenotypic methods, MBL genes were detected in 25 isolates but not in one. This isolate probably harbored a rare MBL gene such as bla DIM (14, 15) that has not been investigated in our study.
In our work, out of 40 carbapenemase-producing P. aeruginosa, 26 isolates were MBL producers by phenotypic test (CDT positive), whereas 14 isolates were CDT negative. It is noteworthy that the bla NDM gene was detected in 7 of these 14 isolates by PCR. Thus, it is probable that these 7 isolates were also MBL producers, but CDT was not able to detect MBL (false negative results).
Based on the results of the phenotypic test, out of 67 isolates that were not susceptible to one or more carbapenems, 26 (38.8%) were MBL producers. The frequency of MBL producers among all 169 isolates using CDT was 15.4% (26/169). In the study performed by Bagheri Bejestani et al. in Tehran, Iran, 3.3% of P. aeruginosa isolates were MBL producers based on CDT results, which was a lower frequency than the frequency obtained from our study (16). In a study conducted by Mirbagheri and colleagues in Mashhad, northeast of Iran, according to CDT results, 88.8% of imipenem-resistant isolates were MBL producers (17). In another study in Ahvaz, southwest of Iran, 90% of imipenem-resistant isolates were phenotypically MBL producers; these researchers also used CDT (1). Therefore, the frequency of MBL-producing isolates in these two studies was higher than the frequency in our study. In an investigation in India carried out by Arunagiri et al.,70.1% of multidrug-resistant P. aeruginosa were MBL producers based on the CDT results (18). In a study in Canada on 241 isolates that were not susceptible to imipenem, 110 (46%) isolates were detected as MBL positive by CDT (5). In Belgium, Heinrichs and colleagues studied 162 multidrug-resistant P. aeruginosa that were not susceptible to imipenem; 52 (32%) isolates were MBL positive in their investigation. Heinrichs et al. used CDT and disk synergy test (DST), but CDT with imipenem showed the best results (12). The reasons for these different results are difficult to explain but may be due to differences in the distribution of MBL genes across various geographical areas, studied isolates, and methods for the phenotypic detection of MBL producers.
In this study, the MIC of imipenem was determined for the 54 imipenem-resistant isolates. The MIC values of imipenem for 53 isolates were ≥32 µg/ml, which indicates a high-level resistance of these 53 isolates to imipenem. Imipenem showed a MIC ≥32 µg/ml for all 26 isolates that were MBL positive by the phenotypic method. In addition, the MIC values of imipenem were ≥32 µg/ml for 9 imipenem-resistant isolates that were MBL negative by phenotypic method but harbored the bla NDM gene.
In this study, bla IMP , bla NDM , and bla VIM genes were detected in 18 (69.2%), 8 (30.8%), and 1 (3.8%) of the MBL-producing isolates, respectively. Among all 169 P. aeruginosa isolates, 23 (13.6%) harbored bla NDM , 18 (10.6%) carried bla IMP , and 1 (0.6%) carried the bla VIM gene. bla SPM , bla GIM , bla AIM , and bla SIM were not found in our work. In a study in Ahvaz, Iran, bla VIM-2 and bla IMP-1 were detected in 1.6% and 55% of imipenem-resistant isolates, respectively, but, no bla SPM-1 gene was found in the isolates (1). In a study done by Haghi and colleagues in Zanjan, northwest of Iran, the frequency of bla IMP , bla VIM , bla SPM , and bla SIM among the MBL-producing isolates was 80%, 17.1%, 57.1%, and 14.1%, respectively. Therefore in contrast to our results, bla SPM and bla SIM were detected in their study. However, similar to our results, no bla GIM harboring isolate was detected. Also, these authors reported that 2 isolates contained bla NDM-1 , bla IMP , and bla SPM (19). Coexistence of bla IMP and bla NDM was also observed in 2 (7.7%) MBL-producing isolates in our work.
In an investigation conducted by Bagheri Bejestani et al. in Tehran, Iran, 3.3% of isolates carried the IMP gene (16) which was lower in frequency compared with our study. In the present study, no bla SPM gene was identified in any of the isolates which was in accordance with the study performed by Yousefi et al. in Tabriz,northwest Iran (20). In the study conducted in Brazil, among the MBL-producing isolates, 55.6% were positive for bla SPM-1 (21).
In an investigation in Iraq, bla NDM was detected in 4 of 22 P. aeruginosa isolates (4). In the study done in India, 17.3% of carbapenem-resistant P. aeruginosa isolates carried bla NDM-1 (22). In our work, among all 169 P. aeruginosa isolates, bla NDM was the most frequently detected MBL gene which emphasizes the need for surveillance and precautions.
It should be noted that in our study bla NDM was identified in 5 isolates that were susceptible to imipenem, meropenem, and doripenem ( Table 3). The reason for susceptibility of these isolates to carbapenems despite the presence of the bla NDM is unclear. These isolates may be silent reservoirs of the bla NDM . A similar report on the bla SPM gene has been published by Pellegrino and colleagues. These authors reported a carbapenem-susceptible P. aeruginosa strain that carried bla SPM ; they stated that such isolates may be silent reservoirs of the bla SPM gene (23).

Conclusion
bla NDM , bla IMP , and bla VIM genes were detected in this study which could be a warning sign about the prevalence of these genes among P. aeruginosa clinical isolates in our region. Proper monitoring and detection of MBL-producing isolates are the essential steps to prevent the spread of these isolates.